I. Field of the Invention
The present invention relates generally to the fields of labeling, radioimaging, radioimmunotherapy, and chemical synthesis. More particularly, it concerns a strategy for radiolabeling target ligands. It further concerns methods of using those radiolabeled ligands to target tumor angiogenesis, hypoxia, apoptosis, disease receptors, disease functional pathways, and disease cell cycles, as well as for the assessment of pharmaceutical agent effectiveness on these biochemical processes.
II. Description of Related Art
Angiogenesis, the proliferation of endothelial and smooth muscle cells to form new blood vessels, is an essential component of the metastatic pathway. These vessels provide the principal route by which certain cells exit the primary tissue site and enter the circulation. For many disease tissue, the vascular density can provide a prognostic indicator of metastatic potential or survival, with highly vascularized tumors having a higher incidence of metastasis than poorly vascularized tissues (Bertolini et al., 1999; Cao, 1999; Smith et al, 2000).
It may be feasible to block angiogenesis and tumor progression by using anti-angiogenic agents. At present, antiangiogenic agents under clinical testing include: naturally occurring inhibitors of angiogenesis (e.g. angiostatin, endostatin, platelet factor-4), (Jiang et al., 2001; Dhanabal et al., 1999; Moulton et al., 1999; Jouan et al., 1999) specific inhibitors of endothelial cell growth (e.g. TNP-470, thalidomide, interleukin-12), (Logothetis et al., 2001; Moreira et al., 1999; Duda et al., 2000) agents neutralizing angiogenic peptides (e.g. antibodies to fibroblast growth factor or vascular endothelial growth factor, suramin and analogues, tecogalan) (Bocci et al., 1999; Sakamoto et al., 1995) or their receptors, (Pedram et al., 2001) agents that interfere with vascular basement membrane and extracellular matrix (e.g. metalloprotease inhibitors, angiostatic steroids), (Lozonschi et al., 1999; Maekawa et al., 1999; Banerjeei et al., 2000) anti-adhesion molecules, (Liao et al., 2000) antibodies such as anti-integrin αvβ3, (Yeh et al., 2001) and miscellaneous drugs that modulate angiogenesis by diverse mechanisms of action (Gasparini 1999).
For example many malignant tumors are angiogenesis-dependent. Several experimental studies suggest that primary tumor growth, invasiveness and metastasis require neovascularization (Sion-Vardy et al., 2001; Guang-Wu et al., 2000; Xiangming et al., 1998). Tumor-associated angiogenesis is a complex, multi-step process under the control of positive and negative soluble factors. Acquisition of the angiogenic phenotype is a common pathway for tumor progression, and active angiogenesis is associated with molecular mechanisms leading to tumor progression (Ugurel et al., 2001). For instance, vascular endothelial growth factor (VEGF) is a mitogen, morphogen and chemoattractant for endothelial cells and, in vivo, is a powerful mediator of vessel permeability (Szus et al., 2000). Interleukin-8 (IL-8) is a chemo-attractant for neutrophils and is a potent angiogenic factor (Petzelbauer et al., 1995). Basic fibroblast growth factor (bFGF) has been associated with tumorigenesis and metastasis in several human cancers (Smith et al., 1999). The prognostic value of angiogenesis factor expression (e.g. VEGF, bFGF, microvessel density, IL-8, MMP-2 and MMP-9) has been determined for cancer patients treated with chemotherapy (Inoue et al., 2000; Burian et al., 1999). These factors regulate metastasis and angiogenesis and may predict the metastatic potential in individual cancer patients (Slaton et al., 2001).
Apoptosis defects in programmed cell death play an important role in tumor pathogenesis. These defects allow neoplastic cells to survive beyond their normal intended lifespan, and subvert the need for exogenous survival factors. Apoptosis defects also provide protection from hypoxia and oxidative stress as the tumor mass expands. They also allow time for genetic alterations that deregulate cell proliferation to accumulate, resulting in interference with differentiation, angiogenesis, and increased cell motility and invasiveness during tumor progression (Reed, 1999). In fact, apoptosis defects are recognized as an important complement to protooncogene activation, as many deregulated oncoproteins that drive cell division also trigger apoptosis (Green and Evan, 2002). Similarly, defects in DNA repair and chromosome segregation normally trigger cell suicide as a defense mechanism for readicating genetically unstable cells. Thus, apoptosis defects permit survival of genetically unstable cells, providing opportunities for selection of progressively aggressive clones (Ionov et al., 2000). Apoptosis defects also facilitate metastasis by allowing epithelial cells to survive in a suspended state, without attachment to extracellular matrix (Frisch and Screaton, 2001). They also promote resistance to the immune system, inasmuch as many of the weapons used for attacking tumors, including cytolytic T cells (CTLs) and natural killer (NK) cells, depend on the integrity of the apoptosis machinery (Tschopp et al., 1999). Finally, cancer-associated defects in apoptosis play a role in chemoresistance and radioresistance, increasing the threshold for cell death and thereby requiring higher doses for tumor killing (Makin and Hickman, 2000). Thus, defective apoptosis regulation is a fundamental aspect of the biology of cancer.
When it comes to the successful eradication of cancer cells by nonsurgical means, all roads ultimately lead to angiogenesis and apoptosis. Essentially all cytotoxic anticancer drugs currently in clinical use block angiogenesis and induce apoptosis of malignant cells. While microtubule binding drugs, DNA-damaging agents, and nucleosides are important weapons in the treatment of cancer, new classes of targeted therapeutics may soon be forthcoming. These new classes of targeted therapeutics may soon be forthcoming based on strategies that have emerged from a deeper understanding of the molecular mechanisms that underlie the phenomenon of angiogenesis and apoptosis (Reed, 2003).
Though angiogenic and apoptotic factors reflect angiogenesis and apoptosis status, these agents may not adequately reflect the therapeutic response of tumors. Currently, methods of assessing angiogenesis and apoptosis in tumors rely on counting microvessel density in the areas of neovascularization and observing annexin V with FACS techniques. After tissue biopsy, immunohistochemistry of tissue specimen is then performed. Both techniques are invasive and cannot be repeatedly performed.
Improvement of scintigraphic tumor imaging is extensively determined by development of more tumor specific radiopharmaceuticals. Due to greater tumor specificity, radiolabeled ligands as well as radiolabeled antibodies have opened a new era in scintigraphic detection of tumors and undergone extensive preclinical development and evaluation (Mathias et al., 1996, 1997a, 1997b). Radionuclide imaging modalities (positron emission tomography, PET; single photon emission computed tomography, SPECT) are diagnostic cross-sectional imaging techniques that map the location and concentration of radionuclide-labeled radiotracers. Although CT and MRI provide considerable anatomic information about the location and the extent of tumors, these imaging modalities cannot adequately differentiate invasive lesions from edema, radiation necrosis, grading or gliosis. PET and SPECT can be used to localize and characterize tumors by measuring metabolic activity.
[18F]FMISO has been used to diagnose head and neck tumors, myocardial infarction, inflammation, and brain ischemia (Martin et al. 1992; Yeh et al. 1994; Yeh et al. 1996; Liu et al. 1994). Tumor to normal tissue uptake ratio was used as a baseline to assess tumor hypoxia (Yet et al. 1996). Although tumor metabolic imaging using [18F]FDG was clearly demonstrated, introducing molecular imaging agents into clinical practice depends on some other factors such as easy availability and isotope cost. [18F]fluorodeoxyglucose (FDG) has been used to diagnose tumors, myocardial infarction, and neurological disease. In addition, PET radiosynthesis must be rapid because of short half-life of the positron isotopes. 18F chemistry is complex and is not reproducible in different molecules.
Several compounds have been labeled with 99mTc using nitrogen and sulfur chelates (Blondeau et al., 1967; Davison et al., 1980). Bis-aminoethanethiol tetradentate ligands, also called diaminodithol compounds, are known to form very stable Tc(V)O complexes on the basis of efficient binding of the oxotechnetium group to two thiol sulfur and two amine nitrogen atoms. Radiometal complexes of 2-pyrrolthiones labeled with 99mTc-2-pyrrolthiones complexes have been developed for use as radiopharmaceuticals for imaging and therapy (WO 0180906A2). 99mTc-L,L-ethylenedicysteine (99mTc-EC) is a recent and successful example of N2S2 chelates. EC can be labeled with 99mTc easily and efficiently with high radiochemical purity and stability, and is excreted through the kidney by active tubular transport (Surma et al., 1994; Van Nerom et al., 1990, 1993; Verbruggen et al., 1990, 1992). Furthermore, 99mTc chelated with ethylenedicysteine (EC) and conjugated with a variety of ligands has been developed for use as an imaging agent for tissue-specific diseases, a prognostic tool or as a tool to deliver therapeutics to specific sites within a mammalian body (WO 0191807A2, AU 0175210A5). 99mTc-EC-chelates have been developed for renal imaging and examination of renal function (U.S. Pat. No. 5,986,074 and U.S. Pat. No. 5,955,053). A method of preparing 99mTc-EC complexes and a kit for performing said method has also been developed (U.S. Pat. No. 5,268,163 and WO 9116076A1).
However, there still exist a need for the development of new agents to target tumor angiogenesis, hypoxia, apoptosis defects, disease receptors, disease functional pathways, and disease cell cycles, as well as for the assessment of the pharmaceutical agent effectiveness on these biochemical processes.